We first showed that nitric oxide (NO), a versatile inflammatory signal, increases prolidase activity by increasing its phosphorylation on serine/threonine residues. In NIH 3T3 mouse fibroblasts stimulated with NO donors (DETA/NO) or in cells transfected with iNOS to activate endogenous NO production, prolidase activity was increased more than 2-fold, without an increase in prolidase protein. Since phosphorylation was a possibility, we immunoprecipitated prolidase and found increased immunodetectible phosphorylated serine/threonine. Since cGMP-PKG pathway is the main mediator of NO effects, we treated cells with 8-bromo-cGMP, a cGMP agonist, and found that it stimulated the phosphorylation of prolidase. Furthermore, the effects of DETA/NO could be blocked by Rp-8-pCPT-cGMP, an inhibitor of cGMP kinase, but not by UO126, an inhibitor of ERK1/2 signaling. Thus, NO activates prolidase activity through phosphorylation of serine/threonine residues mediated by cGMP-PKG signaling and not through MAP kinases. These findings provide a molecular basis for a prolidase response to inflammatory signals. The wound-healing defect in patients with prolidase deficiency has been described as angiopathic in histologic examinations postmortem. This was found both in nonhealing ulcers and at distant organ sites. Since the products of prolidase (proline, hydroxy-proline) are involved in a number of regulatory mechanisms, we asked whether prolidase might play a role in angiogenesis. To test this hypothesis, we obtained a gain-of-function experimental model by transfecting RKO colorectal cancer cells with a prolidase cDNA expression plasmid and isolating stable transfectants which express 15-20 fold that of vector controls as determined by enzyme activity and western blots. The prolidase expressing cells (PL) have increased levels of vascular endothelial growth factor (VEGF) in whole cell extracts by western blots and in conditioned media as measured by ELISA. Glucose transporter-1 (Glut-1) is also increased in PL cells. Since both VEGF and Glut-1 are target genes of HIF-1, we measured HIF-1 transcriptional activity using a hypoxia response element (HRE) luciferase construct and found that HIF-1 was much more active in PL cells. As expected, HIF-1alpha levels were higher in nuclear extracts from PL cells. Since HIF-1alpha is regulated primarily by degradation after prolyl hydroxylation of the oxygen dependent degradation domain (ODD), we used an ODD-luciferase construct to examine the effect of PL on prolyl hydroxylase-dependent proteasomal degradation. We found that ODD degradation was markedly decreased in PL cells. That these mechanisms are due to the catalytic activity of prolidase was shown by the finding that proline and hydroxyproline could augment the effects. Furthermore, inhibition of prolidase activity by N-benzyloxycarbonyl-L-proline (Cbz-Pro) markedly decreased the angiogenic signaling with increased prolidase. In conclusion, accompanying the activation of MMP and increased ECM degradation as a source of stress substrates (proline/hydroxyproline), signals are generated to activate angiogenesis to augment the nutrient supply. We propose that the mechanism for decreased degradation of HIF-1alpha accompanying overexpression of prolidase is due to inhibition of prolyl hydroxylase. Our working hypothesis is that products of prolidase i.e. proline and hydroxyproline, are degraded by proline and hydroxyproline oxidase, respectively to produce pyrroline-5-carboxylate and hydroxy-pyrroline-5-carboxylate. These metabolic intermediates are in tautomeric equilibrium with glutamic-gamma semialdehyde and hydroxy-glutamic-gamma-semialdehyde, respectively. Both of these 5-carbon compunds are similar to alpha-ketoglutarate but their respective gamma carbons are nucleophilic carbonyl groups. We propose that these compounds are inhibitors of prolyl hydroxylase at the binding site for alpha ketoglutarate. Using a pull-down assay for the hydroxylated oxygen-dependent degradation domain, we have preliminary evidence that prolyl hydroxylase activity is inhibited, especially by hydroxy-pyrroline-5-carboxylate